Methods of purifying cannabinoids from plant material

ABSTRACT

The invention relates to methods of preparing cannabinoids in substantially pure form starting from plant material. Also described are substantially pure preparations of various cannabinoids and cannabinoid acids, and also extracts enriched in cannabinoids and cannabinoid acids.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. Å371 of PCTInternational application PCT/GB03/04078, filed Sep. 23, 2003, which waspublished under PCT Article 21(2) in English.

FIELD OF THE INVENTION

The invention relates to methods of preparing cannabinoids insubstantially pure form starting from plant material.

BACKGROUND TO THE INVENTION

Cannabis has been used medicinally for many years, and in Victoriantimes was a widely used component of prescription medicines. It was usedas a hypnotic sedative for the treatment of “hysteria, delirium,epilepsy, nervous insomnia, migraine, pain and dysmenorrhoea”.Historically, cannabis was regarded by many physicians as unique; havingthe ability to counteract pain resistant to opioid analgesics, inconditions such as spinal cord injury, and other forms of neuropathicpain including pain and spasm in multiple sclerosis.

The use of cannabis continued until the middle of the twentieth century,when the recreational use of cannabis prompted legislation whichresulted in the prohibition of its use. The utility of cannabis as aprescription medicine is now being re-evaluated. The discovery ofspecific cannabinoid receptors and new methods of administration havemade it possible to extend the use of cannabis-based medicines tohistoric and novel indications.

The principle cannabinoid components present in herbal cannabis are thecannabinoid acids Δ⁹ tetrahydrocannabinolic acid (Δ⁹ THCA) andcannabidiolic acid (CBDA), with small amounts of the correspondingneutral cannabinoids, respectively Δ⁹ tetrahydrocannabinol (Δ⁹ THC) andcannabidiol (CBD). Cannabidiol was formerly regarded as an inactiveconstituent, however there is emerging evidence that it haspharmacological activity, which is different from that of Δ⁹ THC inseveral respects.

In addition to these major cannabinoids, herbal cannabis may containlower levels of other minor cannabinoids. These may be intermediates inthe biosynthesis of the major cannabinoids and hence exist at only lowlevels in the plant as they are constantly undergoing furtherbiotransformation once they are formed. An example of such a cannabinoidis cannabigerol (CBG). Other minor cannabinoids may represent the endpoint of an alternative biosynthetic pathway to that leading to theformation of the major cannabinoids Δ⁹ THC and CBD. These cannabinoidsare frequently relatively more abundant in the plant, an example beingcannabichromene (CBC).

A special example of a minor cannabinoid that is the end point of abiosynthetic pathway is Δ⁹ Tetrahydrocannabivarin (Δ⁹ THCV). Thiscompound is closely related to Δ⁹ THC, with the only difference instructure being the presence of a propyl (C₃H₇) side chain rather than apentyl (C₅H₁₁) side chain on the aromatic ring. This compound usuallyaccompanies Δ⁹ THC at a level of 1-2% of THC present. However in certainselectively bred varieties of cannabis Δ ⁹ THCV can account for greaterthan 70% of total cannabinoids, with Δ⁹ THC being reduced to the levelof a minor constituent.

Purified forms of certain of the cannabinoids present in herbal cannabisare useful as active pharmaceutical agents. For example, Δ⁹ THC (alsoknown as dronabinol) has been approved by the Food and DrugAdministration (FDA) for the control of nausea and vomiting associatedwith chemotherapy, and also shows potential pharmacological activity inthe treatment of glaucoma, migraine headaches, anxiety, and as ananalgesic. Cannabidiol, formerly regarded as an inactive constituent ofcannabis, has, as aforesaid, itself shown promising pharmacologicalactivity.

In the case of the minor cannabinoids, the difficulties in isolating theminor cannabinoids in a pure state and the absence of commerciallyavailable standards have restricted the investigation of thepharmacology of these compounds and their true therapeutic potential isunknown. Consequently it is of great interest to isolate sufficientlypure samples of these cannabinoids in the quantities required to permitpharmacological studies to be performed.

Purified forms of the cannabinoids and cannabinoid acids are alsopotentially useful as analytical standards, particularly in thecharacterisation of cannabis-derived medicines based on botanical drugsubstances prepared from herbal cannabis.

Thus, there remains a need for purified forms of all of the cannabinoidacids and cannabinoids present in cannabis herb, including the majorcannabinoids Δ⁹ THC and CBD and the minor cannabinoids.

Synthetic forms of certain of the cannabinoids, particularly Δ⁹ THC, CBDand CBN, are commercially available. However, synthetic cannabinoids areextremely expensive. Attention has therefore focussed on thepurification of cannabinoids from plant material.

WO 02/32420 discloses a process for preparing, for example, Δ⁹-THC fromplant material. It utilises CO₂ extraction and ethanol precipitation toobtain “primary extracts” containing Δ⁹-THC and CBD, with reducedamounts of, for example, monoterpenes, sesquiterpenes, hydrocarbons,alkaloids, flavonoids and chlorophylls. The CBD is then converted toΔ⁹-THC by a catalysing reaction. The cannabinoids make up onlyapproximately two-thirds of the composition and are therefore notsubstantially pure.

U.S. Pat. No. 6,403,126 discloses a process in which THC is removed froma cannabis extract using chromatography.

JP 3153625 discloses a method of producing an anti-allergic agent. Inone example, dry seeds of cannabis are subjected to multiple extractionsteps and multiple chromatographic steps.

Biochemical Medicine (1973, vol. 8, P. 341-344) discloses a multi-stepextraction and purification process for producing Δ⁹-THC of unspecifiedpurity.

ODCCP Bulletin on Narcotics (1976, Issue 4) discloses a method ofisolating CBD, THC and CBN using preparative gas chromatography.

U.S. Pat. No. 6,365,416 describes a method of preparing Δ⁹ THC fromplant material which involves extracting the plant material with anon-polar organic solvent, optionally subjecting the extract to a columnchromatography step to produce a residue eluate, subjecting the extractor the residue eluate to a low pressure flash distillation to produce adistillate, optionally subjecting the distillate to a second flashdistillation step, and subjecting the distillate to columnchromatography, normal HPLC or reverse-phase HPLC. The process providesa product containing Δ⁹ THC in an amount greater than 90% by weight.

There remains a need for alternative purification processes which may beused to prepare purified forms of all cannabinoid and cannabinoid acidconstituents of cannabis herb, including the cannabinoid acids Δ⁹ THCAand CBDA, the corresponding free cannabinoids Δ⁹ THC and CBD, and theminor cannabinoids. The present invention relates to such a purificationprocess based on a simple combination of solvent extraction,chromatography and re-crystallisation steps. The process is simple,efficient and economic, and is capable of producing cannabinoids of highpurity, whilst avoiding the need for preparatory HPLC.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a method of obtaining asubstantially pure cannabinoid or cannabinoid acid or a product enrichedin a given cannabinoid or cannabinoid acid comprising:

i) obtaining an extract containing a cannabinoid or cannabinoid acidfrom a plant material;

ii) subjecting the extract of step (i) to a chromatographic step toproduce a partially purified extract;

iii) dissolving the partially purified extract in a first solvent,removing any insoluble material therefrom, and removing the solvent; and

iv) dissolving the product obtained in step iii) in a second solvent,removing any insoluble material therefrom, and removing the solvent toobtain the substantially pure cannabinoid or cannabinoid acid or theproduct enriched in a given cannabinoid or cannabinoid acid, wherein thefirst and second solvents are different, and wherein one of the first orsecond solvents is a solvent which is substantially more polar than thecannabinoid/cannabinoid acid which it is desired to purify, and theother solvent is a solvent which is substantially less polar than thecannabinoid/cannabinoid acid which it is desired to purify.

The method may optionally comprise a further step v) of flashchromatography as an optional further purification step. In the mostpreferred embodiment the flash chromatography step may comprise thefollowing:

v) loading the substantially pure cannabinoid or cannabinoid acid or theproduct enriched in a given cannabinoid or cannabinoid acid onto aChromabond Flash BT 12M silica cartridge column, eluting withhexane:ethyl acetate (98:2) at a flow rate of approximately 5 ml/min.

The invention further relates to substantially pure preparations ofvarious cannabinoids and cannabinoid acids and also products enriched invarious cannabinoids and cannabinoid acids.

DESCRIPTION OF THE INVENTION

The invention relates to a purification process for preparingsubstantially pure cannabinoid or cannabinoid acid or a product enrichedin a given cannabinoid or cannabinoid acid from plant material.

A “substantially pure” preparation of cannabinoid or cannabinoid acid isdefined as a preparation having a chromatographic purity (of the desiredcannabinoid or cannabinoid acid) of greater than 95%, more preferablygreater than 96%, more preferably greater than 97%, more preferablygreater than 98%, more preferably greater than 99% and most preferablygreater than 99.5%, as determined by area normalisation of an HPLCprofile.

The term “product enriched in a given cannabinoid or cannabinoid acid”encompasses preparations having at least 80%, preferable greater than85%, more preferably greater than 90% chromatographic purity for thedesired cannabinoid or cannabinoid acid. Such a product will generallycontain a greater proportion of impurities, non-target materials andother cannabinoids than a “substantially pure” preparation.

The method of the invention may be used to extract/purify cannabinoidsor cannabinoid acids from any plant material known to contain suchcannabinoids or cannabinoid acids. Most typically, but not necessarily,the “plant material” will be derived from one or more cannabis plants.

The term “plant material” encompasses a plant or plant part (e.g. bark,wood, leaves, stems, roots, flowers, fruits, seeds, berries or partsthereof) as well as exudates, and includes material falling within thedefinition of “botanical raw material” in the Guidance for IndustryBotanical Drug Products Draft Guidance, August 2000, US Department ofHealth and Human Services, Food and Drug Administration Centre for DrugEvaluation and Research.

The term “Cannabis plant(s)” encompasses wild type Cannabis sativa andalso variants thereof, including cannabis chemovars (varietiescharacterised by virtue of chemical composition) which naturally containdifferent amounts of the individual cannabinoids, also Cannabis sativasubspecies indica including the variants var. indica and var.kafiristanica, Cannabis indica and also plants which are the result ofgenetic crosses, self-crosses or hybrids thereof. The term “cannabisplant material” is to be interpreted accordingly as encompassing plantmaterial derived from one or more cannabis plants. For the avoidance ofdoubt it is hereby stated that “cannabis plant material” includes herbalcannabis and dried cannabis biomass.

“Decarboxylated cannabis plant material” refers to cannabis plantmaterial which has been subject to a decarboxylation step in order toconvert cannabinoid acids to the corresponding free cannabinoids.

The starting material for the purification process is an extractcontaining a cannabinoid or cannabinoid acid obtained from a plantmaterial.

In a preferred embodiment the “extract containing a cannabinoid orcannabinoid acid” may be a solvent extract of a plant material.Preferred extraction solvents for use in the preparation of this extractinclude non-polar solvents, also alcohols such as ethanol or methanoland liquid carbon dioxide. Preferably the extract is prepared bydissolving plant material in an extraction solvent, removing insolublematerial from the resultant solution (preferably by filtration), andremoving the extraction solvent from the solution (preferably by rotaryevaporation) to form an extract containing a cannabinoid or cannabinoidacid.

Non-polar solvents are particularly preferred for preparing an initialextract from the starting plant material. Any non-polar solvent capableof solubilising cannabinoids or cannabinoid acids may be used. Preferrednon-polar solvents include liquid non-polar solvents comprising lowerC5-C12, preferably C5 to C8, straight chain or branched chain alkanes.The most preferred non-polar solvent for the preparation of freecannabinoids is hexane.

In embodiments wherein the method is to be used for the isolation ofcannabinoid acids then it is preferred to use an acidified extractionsolvent to prepare the initial extract. The primary purpose of thisacidification is to prevent/minimise ionisation of the cannabinoid acid,which could otherwise-adversely affect the purification process. It ispreferred to use acidified non-polar solvents, of the types describedabove. Acidification may be achieved by the additional of a small volumeof acid to the solvent. Generally it is sufficient to add a relativelyweak acid, such as acetic acid. For any given purification process theoptimal amount and type of acid used may be determined empirically. Apreferred acidified solvent is 0.1% acetic-acid in hexane. This is theextraction solvent of choice for preparing an initial extract from thestarting plant material in the preparation of cannabinoid acids.

In embodiments of the method where it is desired to purify freecannabinoids, rather than the cannabinoid acids, the plant material maybe subjected to a decarboxylation step prior to step (i). The purpose ofthe decarboxylation step is to convert cannabinoid acids present in theplant material to the corresponding free cannabinoids. Decarboxylationis preferably carried out by heating the plant material to a definedtemperature for a suitable length of time. Decarboxylation ofcannabinoid acids is a function of time and temperature, thus at highertemperatures a shorter period of time will be taken for completedecarboxylation of a given amount of cannabinoid acid. In selectingappropriate conditions for decarboxylation consideration must, however,be given to minimising thermal degradation of the desirable,pharmacological cannabinoids into undesirable degradation products,particularly thermal degradation of Δ⁹ THC to cannabinol (CBN).

Preferably, decarboxylation is carried out in a multi-step heatingprocess in which the plant material is:

i) heated to a first temperature for a first (relatively short) timeperiod to evaporate off retained water and allow for uniform heating ofthe plant material; and

ii) the temperature is increased to a second temperature for a secondtime period (typically longer than the first time period) until at least95% conversion of the acid cannabinoids to their neutral form hasoccurred.

Preferably the first step is conducted at a temperature in the range offrom 100° C. to 110° C. for 10-20 min. More preferably the firsttemperature is about 105° C. and the first time period is about 15minutes.

Optimum times and temperatures for the second step may vary depending onthe nature of the plant material, and more particularly on thecannabinoid which it is intended to isolate from the plant material, andmay be easily determined by routine experiment. Suitable conditions mayinclude, for example, a temperature in the range of from 115° C. to 125°C. for a time period in the range of from 45 to 75 minutes (typically120° C. for 60 minutes), or a temperature in the range of from 135° C.to 145° C., for a time period in the range of from 15 to 45 minutes.

If the plant material is derived from cannabis plants having a high THCcontent (defined as >90% THC as a percentage of total cannabinoidcontent), the second temperature is preferably in the range of from 115°C. to 125° C., typically 120° C., and the second time period ispreferably in the range of from 45 minutes to 75 minutes, typicallyabout 60 minutes. More preferably the second temperature is in the rangeof from 100° C. to 110° C., typically 105° C., and the second timeperiod is in the range of from 60 to 120 minutes. In another embodiment,most preferred for a mass of plant material greater than 4 kg, thesecond temperature is in the range of from 140° C. to 150° C.,preferably 145° C., and the second time period is in the range of from45 to 55 minutes.

Where the starting “plant material” is freshly harvested or “wet” plantmaterial is may be subjected to a drying step to remove excess moistureprior to step (i). For convenience, decarboxylation and drying may becombined in a single heating step or in a multi-step heating process, asdescribed above.

In a particular embodiment of the method of the invention the “extractcontaining a cannabinoid or cannabinoid acid” prepared from the startingplant material may be a “botanical drug substance” prepared from theplant material, or an ethanolic solution of such a botantical drugsubstance. In the context of this application a “botanical drugsubstance” is an extract derived from plant material, which extractfulfils the definition of “botanical drug substance” provided in theGuidance for Industry Botanical Drug Products Draft Guidance, August2000, US Department of Health and Human Services, Food and DrugAdministration Centre for Drug Evaluation and Research of: “A drugsubstance derived from one or more plants, algae, or macroscopic fungi.It is prepared from botanical raw materials by one or more of thefollowing processes: pulverisation, decoction, expression, aqueousextraction, ethanolic extraction, or other similar processes.”

“Botanical drug substances” derived from cannabis plants include primaryextracts prepared by such processes as, for example, maceration,percolation, and solvent extraction. Solvent extraction may be carriedout using essentially any solvent that dissolvescannabinoids/cannabinoid acids, such as for example C1 to C5 alcohols(e.g. ethanol, methanol), C5-C12 alkanes (e.g. hexane), Norflurane(HFA134a), HFA227 and carbon dioxide. When solvents such as those listedabove are used, the resultant extract typically contains non-specificlipid-soluble material. This can be removed by a variety of processesincluding “winterisation”, which involves chilling to −20° C. followedby filtration to remove waxy ballast, extraction with liquid carbondioxide and by distillation. General protocols for the preparation ofbotanical drug substances from cannabis plant material are described inthe applicant's published International patent application WO 02/064109.

The botanical drug substance is preferably obtained by carbon dioxide(CO₂) extraction followed by a secondary extraction, e.g. an ethanolicprecipitation, to remove a substantial proportion of non-cannabinoidmaterials, e.g. waxes, wax esters and glycerides, unsaturated fatty acidresidues, terpenes, carotenes, and flavenoids and other ballast. Mostpreferably the botanical drug substance is produced by a processcomprising extraction with liquid CO₂, under sub-critical orsuper-critical conditions, and then a further extraction, preferably anethanolic precipitation, to remove significant amounts of ballast.

If it is intended to prepare free cannabinoids from the cannabis plantmaterial then the material is preferably heated to a defined temperaturefor a defined period of time in order to decarboxylate cannabinoid acidsto free cannabinoids prior to extraction of the botanical drugsubstance.

In the most preferred embodiment the botanical drug substance isprepared according to a process comprising the following steps:

i) optional decarboxylation of the plant material,

ii) extraction with liquid CO₂ (most preferably under sub-criticalconditions), to produce a crude botanical drug substance,

iii) precipitation with C1-C5 alcohol to reduce the proportion ofnon-target materials,

iv) removal of the precipitate (preferably by filtration),

v) optional treatment with activated charcoal, and

vi) evaporation to remove C1-C5 alcohol and water, thereby producing afinal botanical drug substance.

A detailed example of such a process is described in the accompanyingExamples.

The “extract containing a cannabinoid or cannabinoid acid” is subjectedto a chromatographic purification step to produce a partially purifiedextract. The purpose of this step is to reduce the amount of“non-target”, i.e. non-cannabinoid or non-cannabinoid acid material, inthe extract and also to provide a degree of separation/fractionation ofthe various cannabinoid/cannabinoid acid components of the crude plantextract obtained in step (i). Typically, the product of thechromatographic step is collected in multiple fractions, which may thenbe tested for the presence of the desired cannabinoid/cannabinoid acidusing any suitable analytical technique (e.g. TLC). Fractions enrichedin the desired cannabinoid/cannabinoid acid may then be selected forfurther purification.

The chromatographic step will preferably comprise column chromatography,and is preferably based on molecular sizing and polarity. Preferredcolumn matrix materials are hydrophilic lipophilic materials, forexample hydroxypropylated cross-linked dextrans such as Sephadex LH-20™.Various different solvents may be used in combination with this type ofmatrix, for example dimethyl sulphoxide, pyridine, water,dimethylformamide, methanol, saline, ethylene dichloride, chloroform,propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol,isopropanol, tetrahydrofuran, dioxane, chloroform/dichloromethane etc.

In the most preferred-embodiment the chromatographic step comprisescolumn chromatography on a Sephadex LH-20™ column, preferably elutingwith a 2:1 mixture of chloroform/dichloromethane. However, any suitablecombination of column packing material and solvent having separationcharacteristics suitable for use in separation (fractionation) ofcannabinoids and cannabinoid acids may be used with equivalent effect.The column eluate is typically collected in several fractions. Thefractions are tested for the presence of the desiredcannabinoid/cannabinoid acid using a suitable analytical technique, andthose fractions containing the highest amounts of the desiredcannabinoid or cannabinoid acid selected for further processing. Solventis then removed from the selected fractions, preferably by rotaryevaporation.

The partially purified product obtained from the chromatographic step isre-dissolved in a first solvent. Any insoluble residues (e.g.particulate material) are removed from the resultant solution, typicallyby filtration. The first solvent is then removed, preferably by rotaryevaporation. The product of this step is re-dissolved in a secondsolvent. Again, any insoluble residues (e.g. particulate material) areremoved from the resultant solution, typically by filtration. The secondsolvent is then removed, preferably by rotary evaporation, to producethe final product, which is a substantial pure cannabinoid orcannabinoid acid or a product enriched in a given cannabinoid orcannabinoid acid.

The purpose of these two “solvent treatment” steps is to removecontaminants, leaving a substantially pure preparation of the desiredcannabinoid or cannabinoid acid.

In the preferred embodiment the first and second solvents are different.One of the first or second solvents is a solvent which is substantiallymore polar than the cannabinoid/cannabinoid acid which it is desired topurify. Treatment with this solvent has the effect of removing unwantedcomponents that are less polar than the desired cannabinoid/cannabinoidacid. The other solvent is a solvent which is substantially less polarthan the cannabinoid/cannabinoid acid which it is desired to purify.Treatment with this solvent has the effect of removing unwantedcomponents that are more polar than the desired cannabinoid/cannabinoidacid. The combined effect of sequential treatment with two such solventsis of “topping and tailing” the partially purified extract to yield asubstantially pure product. The two solvent treatment steps may beperformed in either order. It is immaterial to the overall purificationwhether the “less polar” or “more polar” contaminants are removed first.

The first and second solvents may be essentially any solvents thatdissolve cannabinoids and/or cannabinoid acids and which have thedesired polarity in comparison to the cannabinoid/cannabinoid acid whichit is desired to isolate.

Preferred solvents for use in these treatment steps include alcohols,particularly C1-C5 alcohols, with methanol being particularly preferred,and also C5-C12 straight or branched chain alkanes, most preferablypentane. A particularly preferred combination of first and secondsolvents, which is suitable for use in the preparation of the majorityof cannabinoids and cannabinoid acids, is methanol and pentane. Thesesolvents may be used in either order.

The process of the invention generally results in the isolation ofsubstantially pure cannabinoids or cannabinoid acids of highchromatographic purity. Substantially pure cannabinoids or cannabinoidacids are often obtained as crystalline solids or clear colourlesssolutions. The inventors have determined that the process of theinvention may be used to prepare substantially pure preparations ofcannabinoids or cannabinoid acids having a higher degree ofchromatographic purity than the preparations previously known in theprior art. Therefore, in an extremely important aspect, the process ofthe invention provides a solution to the problem of preparing/isolatingcannabinoids and cannabinoid acids at a high degree of purity. Theprocess is advantageously cheap, amenable to scale-up and applicable toa wide range of different cannabinoids and cannabinoid acids.

The process of the invention may be used to prepare substantially pureforms, or products enriched in, essentially any cannabinoids orcannabinoid acids which occur naturally in plant material (includingfree cannabinoid forms of naturally occurring cannabinoid acids).

The essential features of the process are the same for purification ofall cannabinoids and cannabinoid acids. Cannabis plants generallycontain complex mixtures of cannabinoid acids and cannabinoids, althoughdepending on the variety of cannabis one type of cannabinoid maypre-dominate. The purpose of the chromatographic step (ii) is toseparate the various cannabinoid/cannabinoid acid components of thecrude plant extract obtained in step (i). Typically, the product of thechromatographic step is collected in multiple fractions, which may thenbe tested for the presence of the desired cannabinoid/cannabinoid acidusing any suitable analytical technique (e.g. TLC). Fractions enrichedin the desired cannabinoid/cannabinoid acid may then be selected forfurther purification. Hence, the same simple process steps may beadapted for purification of essentially any plant-derived cannabinoid orcannabinoid acid.

Selectivity for different cannabinoids or cannabinoid acids may beenhanced by selection of appropriate starting plant material. By way ofexample, if it is desired to prepare substantially pure Δ⁹ THC or Δ⁹THCA then “high THC” cannabis plants should preferably be selected asthe starting material. Whereas, if it is desired to preparesubstantially pure CBD or CBDA then “high CBD” cannabis plants shouldpreferably be selected as the starting material. However, it is to beunderstood that the process of the invention is of general utility andis not limited to the use of particular cannabis varieties as thestarting material.

Working with Cannabis plants and cannabinoids may require a Governmentlicense in some territories but Governments generally make such licensesavailable to parties who apply for the purposes of medicinal researchand commercial development of medicines. In the United Kingdom a licensemay be obtained from the Home Office.

The precise cannabinoid content of any particular cannabis plantmaterial may be qualitatively and quantitatively determined usinganalytical techniques well known to those skilled in the art, such asthin-layer chromatography (TLC) or high performance liquidchromatography (HPLC). Thus, one may screen a range of from cannabisplants and select those having a high content of the desired cannabinoidacid or cannabinoid for use as starting material in the process of theinvention.

With the use of conventional selective breeding techniques it ispossible to develop cannabis varieties (chemovars) having varyingcannabinoid content. Using such traditional selective breedingtechniques the inventors have been able to select cannabis varieties(chemovars) having relatively high content of CBD, or of the minorcannabinoids Δ⁹ tetrahydrocannabivarin (Δ⁹ THCV), cannabigerol (CBG) orcannabichromene (CBC). General protocols for growing of medicinalcannabis and for testing the cannabinoid content of cannabis plants aredescribed in the applicant's published International patent applicationWO 02/064109.

Where it is desired to purify free cannabinoids, rather than thecorresponding cannabinoid acids, then the process will generally includea “decarboxylation” step to decarboxylate free cannabinoid acids to thecorresponding free cannabinoid. As aforesaid, a decarboxylation step maybe included prior to step (i) if it is desired to isolate freecannabinoids, or omitted if it is desired to isolate cannabinoid acids.

The process of the invention is particularly preferred for use in thepreparation of substantially pure Δ⁹ tetrahydrocannabinolic acid (Δ⁹THCA), cannabidiolic acid (CBDA), Δ⁹ tetrahydrocannabinol (Δ⁹ THC) andΔ⁹ tetrahydrocannabivarin (Δ⁹ THCV) from cannabis plant material, and inthe preparation of extracts of cannabis plant material highly enrichedin cannabigerol (CBG) or cannabichromene (CBC).

The invention further relates to substantially pure preparations ofcertain cannabinoids and cannabinoids and to products highly enriched incertain cannabinoids.

In particular, the invention provides a substantially pure preparationof Δ⁹ tetrahydrocannabinolic acid (Δ⁹ THCA) having a chromatographicpurity of greater than 95%, more preferably greater than 96%, morepreferably greater than 97% and most preferably greater than 98% by areanormalisation of an HPLC profile. The preparation is typically a paleyellow crystalline solid at room temperature, having a melting point of˜70° C.

The preparation preferably comprises: less than 2%, preferably less than1.5%, most preferably 1% or less Δ⁹ THC (w/w),

less than 2%, more preferably less than 1.5%, more preferably less than1% and most preferably less than 0.5% CBD (w/w),

less than 2%, more preferably less than 1.5%, and most preferably lessthan 1% CBN (w/w).

The inventors are the first to isolate Δ⁹ THCA from plant material atthis level of purity in crystalline form. Pure Δ⁹ THCA is useful as astarting material for the preparation of pure Δ⁹ THC by decarboxylation,also as a chromatographic standard.

The preferred method for preparation of substantially pure Δ⁹ THCA fromcannabis plant material comprises:

i) preparing an extract of the cannabis plant material with 0.1% v/vacetic acid in hexane,

ii) filtering the resultant extract and removing solvent from filtrateby rotary evaporation to form an extract enriched in Δ⁹ THCA,

iii) passing a solution of the resulting Δ⁹ THCA enriched extractthrough a column packed with Sephadex-LH20™, eluting with 2:1chloroform/dichloromethane,

iv) collecting Δ⁹ THCA rich fractions eluted from the column andremoving solvent by rotary evaporation,

v) re-dissolving the crude Δ⁹ THCA obtained in step iv) in methanol,removing insoluble residue by filtration and removing solvent fromfiltrate by rotary evaporation,

vi) re-dissolving the product of step v) in pentane, removing insolubleresidue by filtration and removing solvent from filtrate by rotaryevaporation to produce Δ⁹ THCA crystals.

The cannabis plant material will preferably be derived from cannabisplants having a relatively high Δ⁹ THCA content, most preferablycannabis plants containing >90% Δ⁹ THCA as a percentage of totalcannabinoid content.

The invention further provides a substantially pure preparation ofcannabidiolic acid (CBDA) having a chromatographic purity of greaterthan 90%, more preferable greater than 92% and most preferably greaterthan 94% by area normalisation of an HPLC profile. The preparation istypically a pale yellow crystalline solid at room temperature, having amelting point in the range of from 45-48° C.

The preparation preferably comprises: 5% or less, preferably 4.5% orless, more preferably 4% or less, more preferably 3.5% or less and mostpreferably 3% or less CBD (w/w),

less than 1%, preferably less than 0.8%, more preferably less than 0.6%,more preferably less than 0.4%, more preferably less than 0.2% and mostpreferably less than 0.1% Δ⁹ THCA (w/w),

less than 1%, preferably less than 0.8%, more preferably less than 0.6%,more preferably less than 0.4%, more preferably less than 0.2% and mostpreferably less than 0.1% Δ⁹ THC (w/w).

Again, the inventors are the first to isolate CBDA from plant materialat this level of purity in crystalline form.

The invention further provides a substantially pure preparation ofcannabidiolic acid (CBDA) having a chromatographic purity of greaterthan 94%, more preferably greater than 96% and most preferably greaterthan 98% by area normalisation of an HPLC profile. The preparation ispreferably a clear colourless solution at room temperature.

The preparation typically comprises: 3% or less, more preferably 2% orless, more preferably 1% or less and most preferably 1% or less and mostpreferably less than 0.1% CBD (w/w),

less than 0.8%, more preferably less than 0.6%, more preferably lessthan 0.3% THCA (w/w),

less than 1%, preferably less than 0.8%, more preferably less than 0.6%,more preferably less than 0.4%, more preferably less than 0.2% and mostpreferably less than 0.1% Δ⁹-THC (w/w).

Pure CBDA is useful as a starting material for the preparation of pureCBD by decarboxylation, also as a chromatographic standard and may alsohave pharmaceutical potential. The ability to prepare CBDA at a highlevel of purity will permit further studies of the pharmaceuticalutility of this cannabinoid acid.

The preferred method for preparation of substantially pure CBDA fromcannabis plant material comprises:

i) preparing an extract of the cannabis plant material with 0.1% v/vacetic acid in hexane,

ii) filtering the resultant extract and removing solvent from filtrateby rotary evaporation to form an extract enriched in CBDA,

iii) passing a solution of the resulting CBDA enriched extract through acolumn packed with Sephadex-LH20™, eluting with 2:1chloroform/dichloromethane,

iv) collecting CBDA rich fractions eluted from the column and removingsolvent by rotary evaporation,

v) re-dissolving the crude CBDA obtained in step

iv) in methanol, removing insoluble residue by filtration and removingsolvent from filtrate by rotary evaporation,

vi) re-dissolving the product of step v) in pentane, removing insolubleresidue by filtration and removing solvent from filtrate by rotaryevaporation to produce CBDA crystals or a solution.

The cannabis plant material will preferably be derived from cannabisplants having a relatively high CBDA content, most preferably cannabisplants containing >90% CBDA as a percentage of total cannabinoidcontent.

Where the product of the method is a CBDA solution, the method mayoptionally include a further purification step of flash chromatography,comprising The method may optionally include a further purification stepof flash chromatography comprising vii) loading the substantially puresolution of CBDA onto a Chromabond Flash BT 12M silica cartridge column,eluting with hexane:ethyl acetate (98:2) at a flow rate of approximately5 ml/min.

The invention further provides a substantially pure preparation of Δ⁹tetrahydrocannabinol (Δ⁹ THC) having a chromatographic purity of greaterthan 99% by area normalisation of an HPLC profile. The preparation is asemi-solid at room temperature.

The preparation preferably comprises less 0.5%, preferably than 0.4%,more preferably less than 0.2% and most preferably less than 0.1% CBD(w/w),

less than 0.5%, preferably less than 0.4%, more preferably less than0.2% and most preferably less than 0.1% CBN (w/w).

Most preferably the preparation contains no detectable (<0.1%) CBD andno detectable CBN (<0.1%), as analysed by HPLC.

The inventors are the first to isolate Δ⁹ THC from plant materialat >99% purity and in semi-solid form. Δ⁹ THC has previously beenreported in the literature as a yellow oil and has never been obtainedin crystalline form. The pure Δ⁹ THC is of obvious utility as an activepharmaceutical agent, and is also useful as a chromatographic standard,particularly as a comparative standard in the qualitative analysis ofbotanical drug substances derived from cannabis. The availability ofhighly pure Δ⁹ THC will also facilitate studies of the pharmacology ofΔ⁹ THC.

The preferred method for preparation of substantially pure Δ⁹ THCcomprises:

i) obtaining an ethanolic solution of a botanical drug substance fromdecarboxylated cannabis plant material,

ii) passing the solution obtained in step i) through a column ofactivated charcoal, and collecting the eluate,

iii) remove solvent from the eluate by rotary evaporation to give a Δ⁹THC enriched fraction,

iv) passing a solution of the resulting Δ⁹ THC enriched extract througha column packed with Sephadex LH20, eluting with 2:1chloroform/dichloromethane,

v) collecting Δ⁹ THC rich fractions and removing solvent by rotaryevaporation,

vi) re-dissolving the crude Δ⁹ THC prepared in step v) in methanol,removing insoluble residue by filtration and removing solvent fromfiltrate by rotary evaporation,

vii) re-dissolving the crude Δ⁹ THC prepared in step vi) in pentane,removing insoluble residue by filtration and removing solvent from thefiltrate by rotary evaporation to give a semi-solid preparation of Δ⁹THC.

In this method the ethanolic solution of a botanical drug substance fromdecarboxylated cannabis plant material is preferably obtained by amethod comprising the following steps:

i) harvesting cannabis plant material,

ii) decarboxylation of the plant material,

iii) extraction with liquid carbon dioxide (CO₂), removal of CO₂ torecover crude extract,

iv) dissolution of crude extract in ethanol followed by chilling of thesolution to precipitate unwanted waxes,

v) removal of unwanted waxy material by cold filtration.

The (decarboxylated) cannabis plant material will preferably be derivedfrom cannabis plants having a relatively high THC content, mostpreferably cannabis plants containing >90% THC (Δ⁹ THCA plus Δ⁹ THC) asa percentage of total cannabinoid content. The plant material is subjectto decarboxylation in order to convert the naturally occurring Δ⁹ THCAinto Δ⁹ THC.

The invention still further relates to a substantially pure preparationof Δ⁹ tetrahydrocannabivarin (Δ⁹ THCV) having a chromatographic purityof greater than 95%, more preferable greater than 96%, more preferablegreater than 97%, more preferable greater than 98%, and most preferablegreater than 99% by area normalisation of an HPLC profile. Thepreparation is typically a crystalline solid at room temperature.

The preparation preferably comprises less than 1%, preferably less than0.8%, more preferably less than 0.6%, more preferably less than 0.4%,more preferably less than 0.2% and most preferably less than 0.1% CBD(w/w),

less than 2.0%, preferably less than 1.5%, more preferably less than1.0% and most preferably 0.5% or less Δ⁹ THC (w/w),

less than 1%, preferably less than 0.8%, more preferably less than 0.6%,more preferably less than 0.4%, more preferably less than 0.2% and mostpreferably less than 0.1% CBN (w/w).

Again the inventors are the first to isolate Δ⁹ THCV from plant materialat this level of purity and in crystalline form. The availability ofpure Δ⁹ THCV will permit studies of the pharmacology of this minorcannabinoid and evaluation of its pharmaceutical potential. Pure Δ⁹ THCVis also useful as a chromatographic standard and as a starting materialfor the preparation of pure cannabivarin (CBV), for example by thermaldegradation of Δ⁹ THCV in air.

The preferred method for preparation of substantially pure Δ⁹ THCV fromplant material comprises:

i) obtaining an ethanolic solution of a botanical drug substance fromcannabis plant material,

ii) passing the solution obtained in step i) through a column ofactivated charcoal, and collecting the eluate,

iii) remove solvent from the eluate by rotary evaporation to give a Δ⁹THCV enriched fraction,

iv) passing a solution of the resulting Δ⁹ THCV enriched extract througha column packed with Sephadex LH20, eluting with 2:1chloroform/dichloromethane,

v) collecting Δ⁹ THCV rich fractions and removing solvent by rotaryevaporation,

vi) re-dissolving the crude Δ⁹ THCV prepared in step v) in methanol,removing insoluble residue by filtration and removing solvent fromfiltrate by rotary evaporation,

vii) re-dissolving the crude Δ⁹ THCV prepared in step vi) in pentane,removing insoluble residue by filtration and removing solvent from thefiltrate by rotary evaporation to give crystals of Δ⁹ THCV.

The ethanolic solution of a botanical drug substance from cannabis plantmaterial is preferably obtained by a method comprising the followingsteps:

i) harvesting and decarboxylating cannabis plant material,

ii) extraction with liquid carbon dioxide (CO₂), removal of CO₂ torecover crude extract,

iii) dissolution of crude extract in ethanol followed by chilling of thesolution to precipitate unwanted waxes,

iv) removal of unwanted waxy material by cold filtration.

The cannabis plant material will preferably be derived from cannabisplants having a relatively high Δ⁹ THCV content.

The invention still further comprises a product enriched in cannabigerol(CBG) having a chromatographic purity of greater than 90%, preferablygreater than 92% by area normalisation of an HPLC profile.

The product preferably comprises less than 1%, preferably less than0.8%, more preferably less than 0.6%, more preferably less than 0.4%,more preferably less than 0.2% and most preferably less than 0.1% CBD(w/w),

less than 1%, preferably less than 0.8%, more preferably less than 0.6%,more preferably less than 0.4%, more preferably less than 0.2% and mostpreferably 0.1% or less Δ⁹ THC (w/w).

The product most preferably contains no detectable (<0.1%) CBN or CBDand no more than 0.1% Δ⁹ THC, as analysed by HPLC.

Again, the inventors are the first to prepare cannabis plant extractscontaining the minor cannabinoid CBG at this level of chromatographicpurity.

The invention further provides a substantially pure preparation ofcannabigerol (CBG) having a chromatographic purity of greater than 92%,more preferably greater than 94%, more preferably greater than 96% andmost preferably greater than 97% by area normalisation of an HPLCprofile. The preparation is preferably a clear colourless solution atroom temperature.

The preparation typically comprises: 4% or less, more preferably 3% orless, and most preferably less than 2% CBD (w/w),

less than 1%, preferably less than 0.8%, more preferably less than 0.6%,more preferably less than 0.4%, more preferably less than 0.2% and mostpreferably less than 0.1% Δ⁹-THC (w/w),

less than 1%, preferably less than 0.8%, more preferably less than 0.6%,more preferably less than 0.4%, more preferably less than 0.2%, and mostpreferably less than 0.1% CBN (w/w).

The availability of such enriched extracts or substantially purepreparations will permit further evaluation of the pharmacology of CBGin order to assess its pharmaceutical potential. The enrichedextract/substantially pure preparation is also useful as a referencestandard in chromatographic characterisation of cannabis-derivedmedicines.

The preferred method of preparing enriched CBG extracts or substantiallypure preparations of CBG from cannabis plant material comprises:

i) decarboxylating the cannabis plant material,

ii) preparing an extract of the decarboxylated cannabis plant materialwith hexane,

iii) filtering the resultant extract and removing solvent from filtrateby rotary evaporation to form an extract enriched in CBG,

iv) passing a solution of the resulting CBG enriched extract through acolumn packed with Sephadex-LH20™, eluting with 2:1chloroform/dichloromethane,

v) collecting CBG rich fractions eluted from the column and removingsolvent by rotary evaporation,

vi) re-dissolving the crude CBG obtained in step v) in methanol,removing insoluble residue by filtration and removing solvent fromfiltrate by rotary evaporation,

vii) re-dissolving the product of step vi) in pentane, removinginsoluble residue by filtration and removing solvent from filtrate byrotary evaporation to produce a highly enriched CBG extract orsubstantially pure preparation of CBG.

The cannabis plant material will preferably be derived from cannabisplants having a relatively high CBG content.

Optionally a further step of flash chromatography may be conducted tofurther improve purity, preferably as set out in step viii) below. Sucha step results in a further improvement in purity to greater than 99%(w/w). The skilled person will appreciate that an equivalent step couldbe used to improve purity for any of the other cannabinoids.

Step viii) loading the substantially pure cannabigerol or thecannabigerol enriched product onto a Chromabond Flash BT 12M silicacartridge column, eluting with hexam:ethyl acetate (98:2) at a flow rateof approximately 5 ml/min.

The invention still further comprises a product enriched incannabichromene (CBC) having a chromatographic purity of greater than80%, more preferably greater than 85% by area normalisation of an HPLCprofile.

The product preferably comprises less than 5%, preferably less than 4%,more preferably less than 3%, more preferably less than 2% and mostpreferably 1% or less CBD (w/w),

less than 2%, preferably less than 1.5%, more preferably less than 1.0%,more preferably less than 0.5% and most preferably 0.3% or less Δ⁹ THC(w/w),

less than 1%, preferably less than 0.8%, more preferably less than 0.6%,more preferably less than 0.4%, more preferably less than 0.2% and mostpreferably 0.1% or less CBN (w/w).

Again, the inventors are the first to prepare cannabis plant extractscontaining the minor cannabinoid CBC at this level of chromatographicpurity.

The invention further provides a substantially pure preparation ofcannabichromene (CBC) having a chromatographic purity of greater than85%, more preferably greater than 90%, more preferably greater than 95%,more preferably greater than 98% and most preferably greater than 99% byarea normalisation of an HPLC profile. The preparation is a clearcolourless solution at room temperature.

The preparation typically comprises:

1% or less, more preferably 0.8% or less, more preferably 0.6% or less,more preferably 0.4% or less and most preferably less than 0.2% CBD(w/w),

less than 1%, preferably less than 0.8%, more preferably less than 0.6%,more preferably less than 0.4%, more preferably less than 0.2% and mostpreferably less than 0.1% Δ⁹-THC (w/w),

less than 1%, preferably less than 0.8%, more preferably less than 0.6%,more preferably less than 0.4%, more preferably less than 0.2% and mostpreferably less than 0.1% CBN (w/w).

The availability of such enriched extracts/substantially purepreparations will permit further evaluation of the pharmacology of CBCin order to assess its pharmaceutical potential. The enrichedextract/substantially pure preparation is also useful as a referencestandard in chromatographic characterisation of cannabis-derivedmedicines.

The preferred method for preparing enriched CBC extracts orsubstantially pure preparations of CBC from cannabis plant materialcomprises:

i) decarboxylating the cannabis plant material,

ii) preparing an extract of the decarboxylated cannabis plant materialwith hexane,

iii) filtering the resultant extract and removing solvent from filtrateby rotary evaporation to form an extract enriched in CBC,

iv) passing a solution of the resulting CBC enriched extract through acolumn packed with Sephadex-LH20™, eluting with 2:1chloroform/dichloromethane,

v) collecting CBC rich fractions eluted from the column and removingsolvent by rotary evaporation,

vi) re-dissolving the crude CBC obtained in step v) in methanol,removing insoluble residue by filtration and removing solvent fromfiltrate by rotary evaporation,

vii) re-dissolving the product of step vi) in pentane, removinginsoluble residue by filtration and removing solvent from filtrate byrotary evaporation to produce a highly enriched CBC extract orsubstantially pure preparation.

The cannabis plant material will preferably be derived from cannabisplants having a relatively high CBC content.

The method may optionally include a further purification step of flashchromatography comprising viii) loading the substantially purepreparation of CBC or the product enriched in CBC onto a ChromabondFlash BT 12M silica cartridge column, eluting with hexane:ethyl acetate(98:2) at a flow rate of approximately 5 ml/min.

The invention will be further understood with reference to the followingexperimental examples, together with the accompanying Figures, in which:

FIG. 1 shows TLC a profile of crystalline Δ⁹ THCA, compared to startingmaterial (from G1 cannabis chemovar) and CBD and Δ⁹ THC standards.Chromatographic conditions: SIl G/UV₂₅₄, Mobile phase hexane:diethylether 80:20, double development, Visualisation 0.1% Fast Blue B salt inwater. Standards: 1 mg/ml CBD (BN 10601/C) in MeOH 5 μl applied to TLCplate, 1 mg/ml Δ⁹ THC (BN 10601/B) in MeOH 5 μl applied to TLC plate.Samples: 1 mg/ml THCA starting material in MeOH 5 μl applied to TLCplate, 1 mg/ml crystalline THCA in MeOH 5 μl applied to TLC plate.

FIG. 2 shows HPLC profiles of purified Δ⁹ THCA (98% THCA, 1% THC),compared to starting material (from G1 cannabis chemovar; 72% THCA, 17%THC).

FIG. 3 shows TLC profile of crystalline CBDA, compared to startingmaterial (from G5 cannabis chemovar) and CBD and Δ⁹ THC standards.Chromatographic conditions and standards as for FIG. 1. Samples: 1 mg/mlCBDA starting material in MeOH 5 μl applied to TLC plate, 1 mg/mlcrystalline CBDA in MeOH 5 μl applied to TLC plate.

FIG. 4 shows HPLC profiles of crystalline CBDA (94% CBDA, 3% CBD),compared to starting material (from G5 cannabis chemovar; 72% CBDA, 14%CBD).

FIG. 5 shows an HPLC profile of a colourless solution of CBDA (from G5cannabis chemovar).

FIG. 6 shows TLC profiles of purified Δ⁹ THC compared to BDS startingmaterial and CBD and Δ⁹ THC standards. Chromatographic conditions andstandards as for FIG. 1. Samples: 1 mg/ml Δ⁹ THC starting material inMeOH 5 μl applied to TLC plate, 1 mg/ml purified Δ⁹ THC in MeOH 5 μlapplied to TLC plate.

FIG. 7 shows HPLC profiles of purified Δ⁹ THC (99.6% THC, 0% CBD)compared to starting material (BDS; 89% THC, 2% CBD).

FIG. 8 shows comparative HPLC profiles of purified Δ⁹ THC andcommercially available Δ⁹ THC standard (Sigma; 95% THC, 4% CBN).

FIG. 9 shows GC profiles of purified Δ⁹ THC and starting material (BDS).

FIG. 10 shows TLC profiles of purified Δ⁹ THCV and THCV startingmaterial (BDS) compared to CBD and Δ⁹ THC standards. Chromatographicconditions and standards as for FIG. 1. Samples: 1 mg/ml Δ⁹ THCVstarting material in MeOH 5 μl applied to TLC plate, 1 mg/ml crystallineΔ⁹ THCV in MeOH 5 μl applied to TLC plate.

FIG. 11 shows HPLC profiles of Δ⁹ THCV and starting material (BDS)

FIG. 12 shows GC profiles of purified Δ⁹ THCV and starting material(BDS).

FIG. 13 shows TLC profiles of enriched CBG extract and starting material(BDS from G41 chemovar-decarboxylated) compared to CBD and Δ⁹ THCstandards. Chromatographic conditions and standards as for FIG. 1.Samples: 1 mg/ml CBG starting material in MeOH 5 μl applied to TLCplate, 1 mg/ml enriched CBG extract in MeOH 5 μl applied to TLC plate.

FIG. 14 shows HPLC profiles of enriched CBG extract and startingmaterial (BDS from G41 chemovar-decarboxylated).

FIG. 15 shows an HPLC profile of a colourless solution CBG preparation(from decarboxylated G41 cannabis chemovars).

FIG. 16 shows an HPLC profile of further flash chromatography purifiedCBG preparation compared to improved purity CBG (from decarboxylated G41cannabis chemovars).

FIG. 17 shows GC profiles of enriched CBG extract and starting material(BDS from G41 chemovar-decarboxylated).

FIG. 18 shows TLC profiles of enriched CBC extract and starting material(BDS from G80 chemovar-decarboxylated) compared to CBD and Δ⁹ THCstandards. Chromatographic conditions and standards as for FIG. 1.Samples: 1 mg/ml CBC starting material in MeOH 5 μl applied to TLCplate, 1 mg/ml purified enriched CBC extract in MeOH 5 μl applied to TLCplate.

FIG. 19 shows HPLC profiles of enriched CBC extract and startingmaterial (BDS from G80 chemovar-decarboxylated).

FIG. 20 shows an HPLC profile of a colourless solution CBC preparation(from decarboxylated G80 cannabis chemovars).

FIG. 21 shows GC profiles of enriched CBC extract and starting material(BDS from G80 chemovar-decarboxylated).

EXAMPLES Materials and Methods

Plant Material

GW Pharma Ltd has developed distinct varieties of Cannabis plant hybridsto maximise the output of the specific chemical constituents,cannabinoids. Various types of plant are used; one chemovar, designatedG1 or “high THC” chemovar, produces >90% total cannabinoid content as Δ⁹THC (naturally occurring in the plant in the form of Δ⁹ THCA) and afurther chemovar, designated G5 or “high CBD” chemovar produces >90%total cannabinoid content as CBD (naturally occurring in the plant inthe form of CBDA). Other chemovars yield significant amounts of theminor cannabinoids Δ⁹ THCV (G9 chemovar), CBG (G41 chemovar) and CBC(G80 chemovar). Alternative varieties can be obtained—see for example,Common cannabinoids phenotypes in 350 stocks of cannabis, Small andBeckstead, Lloydia vol 36b, 1973 p 144-156—and bred using techniqueswell known to the skilled man to maximise cannabinoid content.

Solvents

All solvents used in the isolation and analysis of the cannabinoids;n-pentane, hexane, chloroform, dichloromethane, di-ethyl ether,acetonitrile, water, methanol and glacial acetic acid were, unlessotherwise stated, of chromatographic or A.R. grade.

Standards

Reference materials from Sigma were used as standards in the analysis ofextracts, intermediates and finished products, these were: Δ⁹ THC inmethanol BN 10601/B (ca. 0.1 mg/ml) and CBD in methanol BN 10601/C (ca.1 mg/ml).

Solvent Extraction Step

For preparation of Δ⁹ THCA and CBDA samples of G1, THC cannabis chemovar(100 g) and G5, CBD cannabis chemovar (100 g) were extracted twice with0.1% v/v glacial acetic acid in hexane (A.R. grade) at a solvent:herbratio of 15:1. The resulting extracts were filtered and then solventremoved by rotary evaporation to yield crude extracts enriched in therespective cannabinoid acids and suitable for further processing.

For preparation of cannabigerol (CBG) and cannabichromene (CBC) samplesof G41, CBG cannabis chemovar (100 g) and G80, CBC cannabis chemovar(100 g) were decarboxylated at 120° C. for 1 hour and then extractedtwice with hexane at a solvent:herb ratio of 15:1. Following the removalof solvent, this yielded a crude extract enriched in the respectivecompounds CBG and CBC and suitable for further processing.

For preparation of Δ⁹ THC and Δ⁹ THCV ethanolic solutions of botanicaldrug substances were prepared, respectively, from high THC and high THCVcannabis chemovars according to the following process:

harvest cannabis plant material, dry, reduce particle size by milling toless than 2000 μm

↓

for Δ⁹ THC decarboxylate milled plant material by heating toapproximately 105° C. for 15 minutes, followed by approximately 145° C.for minimum of 55 minutes (NB decarboxylation conditions may be varieddepending on nature of target cannabinoid)

↓

extract with liquid carbon dioxide (CO₂) [Food Grade] for up to 10 hoursConditions: Approximately 60 bar±10 bar pressure and 10° C.±5° C.

↓

Removal of CO₂ by depressurisation to recover crude extract

↓

“Winterisation”—Dissolution of crude extract in ethanol followed bychilling solution (−20° C.±5° C./up to 52 hours) to precipitate unwantedwaxes

↓

Removal of unwanted waxy material by cold filtration (20 mm filter)

↓

ethanolic solution of BDS (Stored at −20° C.±5° C.)

Extraction using liquid CO₂ is carried out under sub-critical conditionsat a temperature of approximately 10° C.±5° C. using a pressure ofapproximately 60 bar ±10 bar. Decarboxylated plant material is packedinto a single column and exposed to liquid CO₂ under pressure forapproximately 8 hours, CO₂ mass flow 1250 kg/hr±20%.

Following depressurisation and venting off of the CO₂ the crude BDSextract is collected into sealed vessels. The crude BDS extract is heldat −20° C.±5° C.

The crude BDS extract contains waxes and long chain molecules. Removalis by “winterisation”, whereby the crude BDS extract is warmed to e.g.40° C.±4° C. to liquefy the material. Ethanol is added in the ratio of2:1 ethanol volume to weight of crude BDS extract. The ethanolicsolution is then cooled to −20° C.±5° C. and held at this temperaturefor approximately 48 hours.

On completion of the winterisation the precipitate is removed by coldfiltration through a 20 μm filter, to give an ethanolic solution of theBDS.

Preliminary charcoal clean-up may be carried out by passing theethanolic BDS solution (500 mg/ml) through a disposable plastic column(130 mm×27 mm i.d) packed with activated charcoal (decolourcarb DCL GDCgrade, from Sutcliffe Speakman Carbons, 15.4 g per unit). Absoluteethanol B.P. (Hayman) is used as the solvent.

Ethanol and any water that may be present are removed by evaporation,e.g. rotary evaporation or thin film evaporation under reduced pressure(60° C.±2° C., with vapour at 40° C.±2° C./172 mbar and 72 mbar±4 mbar).The resulting product may be applied directly to the chromatographycolumn.

Column Chromatography Step

Low pressure column chromatography separations were carried out using aglass column (length×internal diameter=1560 mm×24 mm), packed withSephadex LH-20™ (Fluka). The column length:internal diameter ratio wastherefore 65:1. A 2:1 chloroform/dichloromethane mixture was used aseluant. Eluate was collected as 50 ml fractions.

For purification of Δ⁹ THCA and CBDA approximately 20 ml of crudeextract containing the equivalent of 100 g herb was applied to a glasscolumn (dimensions: length 1560 mm×internal diameter 24 mm), packed with400 g of Sephadex LH-20™ stationary phase, as described above. Thequalitative composition of eluted fractions was monitored by TLC.

For the purification of Δ⁹ THC, 2.5 g of charcoal purified BDS (THC)extract was processed through the above low pressure chromatographysystem, (i.e. stationary phase:sample ratio of 160:1). Eluted fractionswere analysed for Δ⁹ THC content by TLC.

For purification of CBG and CBC approximately 20 ml of crude extractcontaining the equivalent of 100 g herb was applied to a glass column(dimensions: length 1560 mm×internal diameter 24 mm), packed with 400 gof sephadex stationary phase.

For the purification of Δ⁹ THCV, 3 g of charcoal purified BDS (THCV)extract was processed through the above low pressure chromatographysystem, (i.e. stationary phase:sample ratio of 133:1).

Solvent Treatment Steps

Steps of re-dissolving extracts in the first and second solvents,filtering to remove insoluble material and removing solvent by rotaryevaporation are carried out according to standard laboratory procedures,such as would be known to those skilled in the art.

TLC Analysis

The qualitative composition of fractions eluted from the chromatographycolumn and other intermediates was monitored by TLC.

TLC uses both retention time and characteristic spot colour toeffectively identify the cannabinoid/cannabinoid acid components in acomplex mixture. Methanolic solutions of the fractions eluted from thechromatographic column are prepared for TLC analysis. An aliquot isspotted onto a TLC plate, alongside suitable reference samples (e.g. forat least Δ⁹ THC and CBD). Following exposure to Fast Blue B reagent, THCand THCA present as pink spots, while CBD and CBDA are orange in colour.Neutrals can be distinguished from the acids by comparison of the Rfvalue to that obtained for the standards. Identity is confirmed bycomparison of Rf and colour of the sample spot, to that obtained for theappropriate standard.

A typical TLC protocol is as follows:

a) Materials and Methods

Equipment:

Application device capable of delivering an accurately controlled volumeof solution i.e. 1 μl capillary pipette or micro litre syringe.

TLC development tank with lid

Hot air blower

Silica gel G TLC plates (SIL N—HR/UV₂₅₄), 200 μm layer with fluorescentindicator on polyester support.

Dipping tank for visualisation reagent.

-   Mobile phase 80% petroleum ether 60:80/20% Diethyl ether.-   Visualisation reagent 0.1% w/v aqueous Fast Blue B salt BN (Sigma    Corp) (100 mg in 100 ml de-ionised water). An optional method is to    scan at UV 254 and 365 nm.    b) Sample Preparation    i) Herbal Raw Material

Approximately 200 mg of finely ground, dried cannabis is weighed into a10 ml volumetric flask. Make up to volume using methanol:chloroform(9:1) extraction solvent.

Extract by ultrasound for 15 minutes. Decant supernatant and usedirectly for chromatography.

ii) Eluted Column Fractions and Intermediate Extracts are Dissolved inMethanol then used Directly. Suitable Dilutions may be DeterminedEmpirically.

iii) Final Products

The final products (pure cannabinoids or enriched extracts) aredissolved in methanol to s suitable concentration (which may bedetermined empirically) then used directly for chromatography. Allsample preparations should produce a final concentration of about 0.5mg/ml.

iv) Botanical Drug Substance

Accurately weigh approximately 50 mg of botanical drug substance into a25 ml volumetric flask. Dissolve to make volume with HPLC grademethanol.

c) Standards

0.1 mg/ml Δ⁹-THC in methanol (Sigma).

0.1 mg/ml CBD in methanol (Sigma).

The standard solutions are stored frozen at −20° C. between uses and areused for up to 12 months after initial preparation.

d) Test Solutions and Method

Apply to points separated by a minimum of 10 mm.

-   i) either 5 μl of herb extract or 1 μl of pure cannabinoid/enriched    extract solution or 1 μl of diluted column eluate as appropriate,-   ii) 5 μl of 0.1 mg/ml Δ⁹-THC in methanol standard solution,-   iii) 5 μl of 0.1 mg/ml CBD in methanol standard solution.

Dry the prepared plate with a hot air blower.

Place the base of the TLC plate in a development tank containing themobile phase and saturated with vapour.

Elute the TLC plate through a distance of 8 cm, then remove the plate.Allow solvent to evaporate from the plate and then repeat the elutionfor a second time (double development). Remove plate and allow it to dryin air.

The entire plate is briefly immersed in the Fast Blue B reagent untilthe characteristic red/orange colour of cannabinoids begins to develop.The plate is removed and allowed to dry under ambient conditions in thedark.

Cannabinoids will give an orange-purple colour:

Cannabidiol CBD orange (fastest running) Δ⁹ Tetrahydrocannabinol THCpink Cannabinol CBN purple Cannabichromene CBC pink purple CannabigerolCBG orange Δ⁹ tetrahydrocannabivarin THCV purple

The corresponding acids form streaks of the same colour as the neutralcomponent spots. The acids run at lower R_(f).

HPLC Analysis

The composition of the isolated products may be determined by HPLCanalysis.

A typical HPLC assay for Δ⁹ THC, Δ⁹ THCA, CBD, CBDA and CBN may becarried out as follows:

a) Materials and Methods

Chromatography Equipment and conditions:

Equipment Agilent (HP) 1100 HPLC system with variable wavelength UVdetector or diode array detector. HPLC Column Discovery C8 5 μm 15 cm ×0.46 cm Pre-Column Kingsorb C18 5 μm 3 cm × 0.46 cm Mobile PhaseAcetonitrile:Methanol:0.25% w/v acetic acid (16:7:6 by volume) ColumnTemp 25° C. Flow Rate 1.0 ml min-1 Detection 220 nm 600 mA f.s.d. Secondwavelength 310 nm Injection Volume 10 μl Run Time 20-25 minutes (may beextended for samples containing small amount of late-eluting peaks)Elution Order CBD, CBDA, Δ⁹ THCV, CBN, Δ⁹ THC, CBC, Δ⁹ THCAb) Sample Preparation

Samples of “pure” cannabinoids/cannabinoid acids and enriched extractsare diluted in methanol prior to HPLC analysis. Optimal dilutions may bedetermined empirically.

Herbal cannabis samples are prepared by taking a 100 mg sample andtreating this with 5 or 10 ml of Methanol/Chloroform (9/1 w/v). Thedispersion is sonicated in a sealed tube for 10 minutes, allowed to cooland an aliquot is centrifuged and suitably diluted with methanol priorto chromatography.

c) Standards

Stock standard solutions of CBD, CBN and Δ⁹ THC in methanol atapproximately 1 mg ml-1 are stored at −20° C. Diluted working standards(0.1 mg/ml for Δ⁹ THC and CBD and 0.01 mg/ml for CBN) are prepared inmethanol from the stock standards and stored at −20° C. (maximum periodof twelve months after initial preparation). After preparation, standardsolutions must be aliquoted into vials to reduce the amount of standardexposed to room temperature. Prior to use in an HPLC sample assay, therequired number of standard vials are removed and allowed to equilibrateto room temperature.

Injection of each standard is made in triplicate prior to the injectionof any test solution. At suitable intervals during the processing oftest solutions, repeat injections of standards are made. In the absenceof reliable CBDA and Δ⁹ THCA standards, these compounds are analysedusing respectively the CBD and Δ⁹ THC standard response factors.

d) Test Solutions

Diluted test solutions are made up in methanol and should containanalytes in the linear working range of from 0.02-0.2 mg/ml.

e) Chromatography Acceptance Criteria:

The following acceptance criteria are applied to the results of eachsequence as they have been found to result in adequate resolution of allanalytes (including the two most closely eluting analytes CBD and CBDA)

TABLE 1 Retention time windows and Relative Retention Time (RRT) to Δ⁹THC for each analyte Retention time Cannabinoid (minutes) RRT (THC) CBD5.1-5.8 0.58 CBN 7.4-8.3 0.83 Δ⁹ THC  9.0-10.0 1.00 CBDA 5.5-6.2 0.615Δ⁹ THCV 5.9-6.2 0.645 CBC 11.6-12.8 1.30 Δ⁹ THCA 14.6-16.0 1.605

TABLE 2 Peak Shape (Symmetry Factor according to British Pharmacopoeiamethod) Cannabinoid Symmetry factor CBD <1.30 CBN <1.25 Δ⁹ THC <1.35f) Data Processing

Cannabinoids can be subdivided into neutral and acidic the qualitativeidentification can be performed using the DAD dual wavelength mode.Acidic cannabinoids absorb strongly in the region of 220 nm-310 nm.Neutral cannabinoids only absorb strongly in the region of 220 nm.

Routinely, only the data recorded at 220 nm is used for quantitativeanalysis.

The DAD can also be set up to take UV spectral scans of each peak, whichcan then be stored in a spectral library and used for identificationpurposes.

Data processing for quantitation utilises batch processing software onthe Hewlett Packard Chemstation.

g) Calculation:

Chromatographic purity of cannabinoid samples is calculated as a % oftotal cannabinoid content by area normalization.

Capillary Gas Chromatography (GC) Analysis

a) Chromatography Equipment and Conditions

Equipment Agilent (HP) 5890 or 6890 GLC system with HP7673 Autosamplerand FID detector GLC column SE54 (EC5) 30 m × 0.32 mm i.d. (Alltech)phase thickness 0.25 μm Flow rate Constant pressure (10.3 psi). Normalinitial flow rate 34 cm sec⁻¹ (2.0 ml min⁻¹) Column oven 70° C.initially then ramp 5° C. min⁻¹ to 250° C. Hold at 250° C. for 15minutes. Injector temp 250° C. Detector temp 325° C. Injection Vol 1 μl,split ratio 2.5:1 Run time 45 minutes Fuel gases Hydrogen 40 ml min⁻¹Air 450 ml min⁻¹ Helium 45 ml min⁻¹b) Standard Preparation

Stock standard solutions of CBD, CBN and Δ⁹ THC in methanol atapproximately 1 mg ml-1 are stored at −20° C. Diluted working standards(0.1 mg/ml for Δ⁹ THC and CBD and 0.01 mg/ml for CBN) are prepared inmethanol from the stock standards and stored at −20° C. (maximum periodof twelve months after initial preparation). Allow an aliquot pipettedinto an autosampler vial to equilibriate to room temperature prior touse in a GC assay.

c) Sample Preparation

Samples of final products, i.e. “pure” cannabinoids/cannabinoid acidsand enriched extracts are diluted in methanol prior to HPLC analysis.Optimal dilutions may be determined empirically.

Cannabis plant material samples are prepared by taking 100 mg choppeddried material and treating this with 5 or 10 ml of Methanol/Chloroform(9:1 v/v). Extract the sample in an ultrasonic bath for 15 minutes andallow to stand in the dark for 18 hours.

d) Chromatography Procedure

Standard solutions are used to provide quantitative and retention timedata. These can be typically injected in triplicate prior to theinjection of any sample solutions and then singularly at suitableintervals during the run, with a maximum of 10 test samples in betweenstandards.

TABLE 3 Retention times THCV 33.7-34.5 minutes CBD 35.6-36.3 minutes Δ⁹THC 37.2-38.1 minutes CBN 38.5-39.1 minutes

Example 1 Preparation of Δ⁹ THCA

Summary of Process:

Extract THC herb (G1 chemovar) with 0.1% v/v acetic acid in hexane.

↓

Filter and remove solvent from filtrate on rotary evaporator.

↓

Pass a solution of the resulting THCA enriched extract through a columnpacked with Sephadex LH₂O, eluting with 2:1 chloroform/dichloromethane.

↓

Collect THCA rich fractions and remove solvent by rotary evaporation.

↓

Re-dissolve crude THCA in methanol and remove insoluble residue byfiltration.

↓

Remove solvent from filtrate by rotary evaporation.

↓

Re-dissolve crude THCA in pentane and remove insoluble residue byfiltration.

↓

Remove solvent from filtrate by rotary evaporation.

↓

Δ⁹ THCA crystals

Results:

Yield:

100 g of G1 chemovar yields approx 5 g of purified Δ⁹ THCA

Characteristics:

Pale yellow crystalline solid.

Chromatographic purity=98% by area normalization.

CBD<0.5% w/w

THC=1.0% w/w

CBN<1.0% w/w

Melting point=70° C. (with decomposition).

Material slowly decarboxylates in solutionΔ⁹ THCA→Δ⁹ THC+CO₂

Example 2 Preparation of CBDA

Summary of Process:

Extract CBD herb (G5 chemovar) with 0.1% v/v acetic acid in hexane.

↓

Filter and remove solvent from filtrate on rotary evaporator.

↓

Pass a solution of the resulting CBDA enriched extract through a columnpacked with Sephadex LH20, eluting with 2:1 chloroform/dichloromethane.

↓

Collect CBDA rich fractions and remove solvent by rotary evaporation.

↓

Re-dissolve crude CBDA in methanol and remove insoluble residue byfiltration.

↓

Remove solvent from filtrate by rotary evaporation.

↓

Re-dissolve crude CBDA in pentane and remove insoluble residue byfiltration.

↓

Remove solvent from filtrate by rotary evaporation.

↓

i) CBDA crystals or ii) CBDA solution

For i) above:

Yield:

100 g of G5 chemovar yields approx 5 g of purified CBDA.

Characteristics:

Pale yellow crystalline solid

Melting Point=45-48° C.

Chromatographic purity=94% CBDA by area normalisation with reference toFIG. 4

*CBD 3%.

THCA non detected i.e. <0.1%

THC non detected i.e. <0.1% * As CBDA does not co-elute with CBD duringprocessing of the extract in the low pressure column chromatographymethod employed, the detected CBD is likely to be formed from thebreakdown of the CBDA during processing and analysis. This undesirabledecarboxylation of the purified material might be minimised bymanipulation of CBDA at sub-ambient temperatures.

Material slowly decarboxylates in solutionCBDA→CBD+CO₂For ii) above:Characteristics:

Clear colourless solution

Chromatographic purity=98.9% CBDA by area normalisation with referenceto FIG. 5

THCA 0.28%

Example 3 Preparation of Δ⁹ THC

Summary of Process:

Ethanolic solution of BDS (approx 400 mg/ml) passed through a column ofactivated charcoal, and eluate collected.

↓

Remove solvent by rotary evaporation to give THC enriched fraction.

↓

Pass a solution of the resulting THC enriched extract through a columnpacked with Sephadex LH₂O, eluting with 2:1 chloroform/dichloromethane.

↓

Collect THC rich fractions and remove solvent by rotary evaporation.

↓

Re-dissolve crude THC in methanol and remove insoluble residue byfiltration.

↓

Remove solvent from filtrate by rotary evaporation.

↓

Re-dissolve crude THC in pentane and remove insoluble residue byfiltration.

↓

Remove solvent from filtrate by rotary evaporation.

↓

Δ⁹ THC SEMI-SOLID

Yield:

3.5 g of Δ⁹ THC BDS yields approx 1.5 g of purified Δ⁹ THC.

Characteristics:

Clear semi-solid which rapidly takes on a purple colour when exposed toair.

(This colour change is reversible when the material is redissolved in asuitable solvent).

Chromatographic purity>99% Δ⁹ THC by area normalization.

Chromatographic purity superior to commercially available Δ⁹ THC Sigmastandard

CBD non detected i.e. <0.1%

CBN non detected i.e. <0.1%

Identity confirmed by HPLC, GC and TLC retention behaviour compared toΔ⁹ THC Sigma standard.

Example 4 Preparation of Δ⁹ THCV

Summary of Process:

Ethanolic solution of BDS, derived from G9 chemovar, passed throughcolumn of activated charcoal, and eluate collected.

↓

Remove solvent by rotary evaporation to give enriched cannabinoidextract.

↓

Pass a solution of the resulting concentrated extract through a columnpacked with Sephadex LH₂O and eluting with 2:1chloroform/dichloromethane.

↓

Collect THCV rich fractions and remove solvent by rotary evaporation.

↓

Re-dissolve crude THCV enriched fractions in methanol and removeinsoluble residue by filtration.

↓

Remove solvent from filtrate by rotary evaporation.

↓

Re-dissolve crude THCV enriched fractions in pentane and removeinsoluble residue by filtration.

↓

Remove solvent from filtrate by rotary evaporation.

↓

Crystalline THCV

Yield:

4.0 g of Δ⁹ THCV BDS yields approx 1.3 g of purified Δ⁹ THCV.

Characteristics:

Off white crystals which rapidly take on a purple colour when exposed toair. This colour change is reversible when the crystals are redissolved.

Chromatographic purity >99% by area normalization.

CBD non detected i.e. <0.1%

THC 0.5%

CBN non detected i.e. <0.1%

Superior to BDS THCV, which contains 75% THCV & 17% THC as % of totalcannabinoids, for studies of chemistry and pharmacology of THCV.

Identity confirmed by HPLC & GC retention times versus THCV fractionpreviously authenticated by GC-MS.

Example 5 Preparation of Cannabigerol (CBG)

Summary of Process:

Extract decarboxylated G41 chemovar with hexane.

↓

Filter and remove solvent from filtrate on rotary evaporator.

↓

Pass a solution of the resulting concentrated extract through a columnpacked with Sephadex LH₂O and eluting with 2:1chloroform/dichloromethane.

↓

Collect CBG rich fractions and remove solvent by rotary evaporation.

↓

Re-dissolve crude CBG enriched fractions in methanol and removeinsoluble residue by filtration.

↓

Remove solvent from filtrate by rotary evaporation.

↓

Re-dissolve crude CBG enriched fractions in pentane and remove insolubleresidue by filtration.

↓

Remove solvent from filtrate by rotary evaporation.

↓

i) Highly enriched CBG extract or ii) CBG solution

↓

Flash chromatography

For i) above:

Yield:

100 g of G41 chemovar yields approx 300 mg of CBG enriched fraction.

Characteristics:

Orange/yellow semi-solid.

Identification by GC retention index relative to THC & CBD standards[ref: Brenneisen, R. & El Sohly, M. A., “Chromatographic & spectroscopicProfiles of Cannabis of Different Origins: Part I,” Journal of ForensicSciences, JFSCA, vol. 33, No. 6, pp. 1385-1404, 1988].

Chromatographic purity >92% by area normalization with reference to FIG.14.

CBD non-detected i.e. <0.1%

THC 0.1%

CBN non-detected i.e. <0.1%

For ii) above:

Characteristics:

Clear colourless solution

Chromatographic purity=97.2% CBG by area normalisation with reference toFIG. 15

CBD 1.66%

CBN non-detected i.e. <0.1%

Following flash chromatography of product ii):

Characteristics:

Clear colourless solution

Chromatographic purity=99.9% CBG by area normalisation with reference toFIG. 16

Example 6 Preparation of Cannabichromene (CBC)

Summary of Process:

Extract decarboxylated G80 chemovar with hexane.

↓

Filter and remove solvent from filtrate on rotary evaporator.

↓

Pass a solution of the resulting concentrated extract through a columnpacked with Sephadex LH₂O and eluting with 2:1chloroform/dichloromethane.

↓

Collect CBC rich fractions and remove solvent by rotary evaporation.

↓

Re-dissolve crude CBC enriched fractions in methanol and removeinsoluble residue by filtration.

↓

Remove solvent from filtrate by rotary evaporation.

↓

Re-dissolve crude CBC enriched fractions in pentane and remove insolubleresidue by filtration.

↓

Remove solvent from filtrate by rotary evaporation.

↓

i) Highly enriched CBC extract or ii) CBC solution

For i) above:

Yield:

100 g of G80 chemovar yields approx 300 mg of CBC enriched fraction.

Characteristics:

Orange/yellow semi-solid.

Identification by GC retention index relative to THC & CBD standards[ref: Brenneisen, R. & El Sohly, M. A., “Chromatographic & spectroscopicProfiles of Cannabis of Different Origins: Part I,” Journal of ForensicSciences, JFSCA, vol. 33, No. 6, pp. 1385-1404, 1988].

Chromatographic purity >85% by area normalization with reference to FIG.19.

CBD 1.0%

THC 0.3%

CBN 0.1%

For ii) above:

Characteristics:

Clear colourless solution

Chromatographic purity=99.6% CBC by area normalisation with reference toFIG. 20

CBD 0.12%

CBN 0.09%

1. A method for producing Δ⁹ tetrahydrocannabivarin (Δ⁹ THCV) crystalscomprising: i) obtaining an ethanolic solution of a botanical drugsubstance from cannabis plant material, ii) passing the solutionobtained in step i) through a column of activated charcoal, andcollecting the eluate, iii) remove solvent from the eluate by rotaryevaporation to give a Δ⁹ THCV enriched fraction, iv) passing a solutionof the resulting Δ⁹ THCV enriched extract through a column packed withSephadex LH20, eluting with 2:1 chloroform/dichloromethane, v)collecting Δ⁹ THCV rich fractions and removing solvent by rotaryevaporation, vi) re-dissolving the crude Δ⁹ THCV prepared in step v) inmethanol, removing insoluble residue by filtration and removing solventfrom the filtrate by rotary evaporation, and vii) re-dissolving thecrude Δ⁹ THCV prepared in step vi) in pentane, removing insolubleresidue by filtration and removing solvent from the filtrate by rotaryevaporation to give said crystals of Δ⁹ THCV.
 2. A substantially purepreparation of Δ⁹ tetrahydrocannabivarin (Δ⁹ THCV) obtained by themethod of claim 1, having a chromatographic purity of greater than 95%,more preferably greater than 96%, more preferably greater than 97%, morepreferably greater than 98%, and most preferably greater than 99% byarea normalisation of an HPLC profile.